Method for purifying pluri-differentiated mesenchymal progenitor cells

ABSTRACT

There is provided an isolated pluri-differentiated human mesenchymal progenitor cells (MPCs), a method for isolating and purifying human mesenchymal progenitor cells from Dexter-type cultures, and characterization of and uses, particularly therapeutic uses for such cells. Specifically, there is provided isolated MPCs which can be used for diagnostic purposes, to enhance the engraftment of hematopoietic progenitor cells, enhance bone marrow transplantation, or aid in the treatment or prevention of graft versus host disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/055,292, filed Feb. 10, 2005, which is a continuation of U.S.application Ser. No. 09/914,508, filed Nov. 7, 2001, now U.S. Pat. No.6,936,281, issued Aug. 30, 2005, which is the National Stage ofInternational Application Number PCT/US01/16408, filed May 21, 2001,which claims benefit of U.S. Provisional Application Ser. Nos.60/277,700, filed Mar. 21, 2001, and 60/209,245, filed Jun. 5, 2000,each of which is hereby incorporated by reference herein in itsentirety, including any figures, tables, nucleic acid sequences, aminoacid sequences, and drawings.

GOVERNMENT SUPPORT

The subject matter of this application has been supported by a researchgrant from the National Institutes of Health under grant number R29 HL59683. Accordingly, the government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to pluri-differentiated mesenchymalprogenitor cells that are isolated from hematopoietic cells andmacrophages in Dexter-type cultures and therapeutic uses for such cells.More specifically, the isolated mesenchymal progenitor cells can be usedfor diagnostic purposes, to enhance engraftment of human bone marrow orhematopoietic progenitor cells, or to treat graft versus host disease.

BACKGROUND OF THE INVENTION

Bone marrow, the site of blood cell production and home to variousleukemia and lymphoma cells, comprises a complex cellular populationincluding hematopoietic progenitor or stem cells and the stromal cellsthat support them. Hematopoietic stem cells have the capacity forself-regeneration and for generating all blood cell lineages whilestromal stem cells have the capacity for self-renewal and for producingthe hematopoietic microenvironment.

Two bone-marrow culture systems introduced in the mid-1970's haveevolved as favored media for the in vitro analysis of mesengenesis andhematopoiesis. The Friedenstein culture system was introduced in 1976 asa media for the analysis and study of mesengenesis (Friedenstein et al.Exp Hematol, 1976, 4:267-274). In order to obtain mesenchymal stem cells(MSCs) for expansion in the culture medium, it is necessary to firstisolate rare pluripotant mesenchymal stem cells from other cells in thebone marrow. In the Friedenstein culture system, isolating thenonhematopoietic cells is achieved by utilizing their tendency to adhereto plastic. Once isolated, a monolayer of homogeneous, undifferentiatedstromal cells is then grown in the culture medium, in the absence ofhematopoietic cells. The stromal cells from this system have thepotential to differentiate into discrete mesenchymal tissues, namelybone, cartilage, adipose tissue and muscle depending on specific growthsupplements. These MSCs have been the target of extensive investigationincluding exploration of their potential clinical utility in repair orreplacement of genetically damaged mesenchymal tissues.

In 1977, Dexter et al. developed another bone marrow culture system forthe study of hematopoiesis (Dexter et al. J Cell Physiol, 1977,91:335-344). The Dexter culture does not require isolation of themesenchymal cells before culturing, thus the monolayer of stromal cellsis grown in the presence of hematopoietic cells. Greenberger latermodified the Dexter system by the addition of hydrocortisone to theculture medium, making it more reproducible (Greenberger, Nature, 1978,275:752-754).

Based on the Dexter system's ability to support sustained growth andpreservation of hematopoietic progenitor cells, it has become thestandard in vitro model for the study of hematopoiesis. Although theDexter-type stromal cells and the MSCs in Friedenstein-type culturesexpress similar cytokine/growth factor profiles, the Dexter cultureshave been found to be more efficient at maintaining preservation ofhematopoietic progenitor cells. Over the last 23 years, questions haveremained as to whether the cells from the Dexter cultures retained thepotential to differentiate, like the MSCs in the Friedenstein culture,or whether they have differentiated into another and discrete phenotypedue to their interaction with the hematopoietic cells (Prockop, Science,April 1997, 276(5309):71-74). It has been widely believed that thestromal cells of the Dexter cultures are a heterogeneous mixture ofadipocytes, osteoblasts, fibroblasts, muscle cells, and vascularendothelial cells.

The in vitro analysis and study of hematopoiesis in Friedenstein andDexter culture systems has been of great importance in both veterinaryand human medicine. A number of diseases and immune disorders, as wellas malignancies, appear to be related to disruptions within thehematopoietic system.

Allogeneic bone marrow transplantation is the preferred treatment for avariety of malignant and genetic diseases of the blood and blood-formingcells. The success rate of allogeneic bone marrow transplantation is, inlarge part, dependent on the ability to closely match the majorhistocompatibility complex of the donor cells with that of the recipientcells to minimize the antigenic differences between the donor and therecipient, thereby reducing the frequency of host-versus-graft responsesand graft-versus-host disease (GvHD). Unfortunately, only about 20% ofall potential candidates for bone marrow transplantation have a suitablefamily member match.

Bone marrow transplantation can be offered to those patients who lack anappropriate sibling donor by using bone marrow from antigenicallymatched, genetically unrelated donors (identified through a nationalregistry), or by using bone marrow from a genetically related sibling orparent whose transplantation antigens differ by one to three of sixhuman leukocyte antigens from those of the patient. Unfortunately, thelikelihood of fatal GvHD and/or graft rejection increases from 20% formatched sibling donors to 50% in the cases of matched, unrelated donorsand un-matched donors from the patient's family.

The potential benefits of bone marrow transplantation have stimulatedresearch on the cause and prevention of GvHD. The removal of T cellsfrom the bone marrow obtained from matched unrelated or unmatchedsibling donors results in a decreased incidence of graft versus hostreactions, but an increased incidence of rejection of the allogeneicbone marrow graft by the patient.

Current therapy for GvHD is imperfect, and the disease can bedisfiguring and/or lethal. Thus, risk of GvHD restricts the use of bonemarrow transplantation to patients with otherwise fatal diseases, suchas severe immunodeficiency disorders, severe aplastic anemia, andmalignancies.

The potential to enhance engraftment of bone marrow or stem cells fromantigenically mismatched donors to patients without graft rejection orGvHD would greatly extend the availability of bone marrowtransplantation to those patients without an antigenically matchedsibling donor.

Thus, it would be useful to develop methods of improving bone marrowtransplantation by enhancing the engraftment of bone marrow orhematopoietic progenitor cells and/or decreasing the occurrence of graftrejection or GvHD in allogenic transplants.

Studies of hematopoiesis and mesengenesis and the urgent need forimproved methods of treatment in the field of bone marrow transplantshave led to the isolation of MSCs from bone marrow stroma. These MSCsare the same pluri-potential cells that result from expansion inFriedenstein type cultures. Several patents describe the isolation andtherapeutic uses of these MSCs.

U.S. Pat. No. 5,486,359 to Caplan et al. is directed to isolated humanMSCs, and a method for their isolation, purification, and culturing.Caplan et al. also describes methods for characterizing and using thepurified mesenchymal stem cells for research, diagnostic, andtherapeutic purposes. The invention in '359 to Caplan et al. describespluri-potential cells that remain pluri-potential, even after culturalexpansion. Caplan et al. also teaches that it is necessary to firstisolate the pluri-potent MSCs from other cells in the bone marrow andthen, in some applications, uses culture medium to expand the populationof the isolated MSCs. This patent fails to disclose the use ofDexter-type cultures, pluri-differentiated mesenchymal progenitor cells,or the isolation of cells from Dexter-type cultures.

U.S. Pat. No. 5,733,542 to Haynesworth et al., is directed to methodsand preparations for enhancing bone marrow engraftment in an individualby administering culturally expanded MSC preparations and a bone marrowgraft. U.S. Pat. No. 6,010,696 to Caplan et al., is directed to methodsand preparations for enhancing hematopoietic progenitor cell engraftmentin an individual by administering culturally expanded MSC preparationsand hematopoietic progenitor cells. The cells utilized in Haynesworth etal. and '696 to Caplan, et al. are the pluri-potential cells describedin U.S. Pat. No. 5,486,359, above. Neither patent discloses the use ofDexter-type cultures, pluri-differentiated mesenchymal progenitor cells,or the isolation of cells from Dexter-type cultures.

Mesenchymal stem cells that are isolated from bone marrow are furtherdescribed by Prockop (Science, 1997, 276(5309):71-4) and Pittenger etal. (Science, 1999, 284(5411):143-147). These articles also describepluri-potential but undifferentiated MSCs and fail to teach or disclosea pluri-differentiated mesenchymal cell or the isolation of mesenchymalcells from Dexter-type cultures.

While they may provide some benefit, the isolated MSCs in the prior arthave not solved the problems associated with engraftment ofhematopoietic progenitor cells or bone marrow engraftment. Consequently,there exists a need in the art for methods of improving engraftment ofhematopoietic progenitor cells and engraftment of bone marrow in mammalsin need of such treatment. There also exists a need in the art fortreating and preventing the occurrence of GvHD in mammals that receiveallogeneic bone marrow transplants.

BRIEF SUMMARY OF THE INVENTION

According to the present invention there is provided isolatedpluri-differentiated mesenchymal progenitor cells, a method ofisolation, diagnostic uses, and therapeutic uses relating to enhancingthe engraftment of human bone marrow or hematopoietic progenitor cellsand treating GvHD.

The present invention provides an isolated mesenchymal progenitor cellthat is pluri-differentiated.

Accordingly, the present invention also provides a method for purifyingpluri-differentiated mesenchymal progenitor cells including the stepsof: providing a cell culture preparation by the Dexter method, treatingthe cells to obtain a cell suspension, removing macrophages,fractionating the cells, and collecting the fraction ofpluri-differentiated mesenchymal progenitor cells.

The present invention also provides a method for enhancing bone marrowengraftment in a mammal in need thereof which includes administering tothe mammal (i) isolated pluri-differentiated mesenchymal progenitorcells and (ii) a bone marrow graft, wherein the pluri-differentiatedmesenchymal progenitor cells are administered in an amount effective topromote engraftment of the bone marrow in the mammal.

The present invention provides a method for enhancing engraftment ofhematopoietic progenitor cells in a mammal in need thereof whichincludes the step of administering to the mammal (i) isolatedpluri-differentiated mesenchymal progenitor cells and (ii) hematopoieticprogenitor cells, wherein the pluri-differentiated mesenchymalprogenitor cells are administered in an amount effective to promoteengraftment of the hematopoietic progenitor cells in the mammal.

Another embodiment of the present invention provides a method fortreating graft-versus-host disease (GvHD) in a mammal about to undergobone marrow or organ transplantation or suffering from GvHD caused bybone marrow or organ transplantation, by administering to the mammal aneffective amount of isolated pluri-differentiated mesenchymal progenitorcells.

Yet another embodiment of the present invention provides a method fordiagnosing a disease state by: a) establishing gene expression patternsof normal state bone marrow derived isolated pluri-differentiatedmesenchymal progenitor cells; b) establishing gene expression patternsof various leukemic state bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cells; c) identifying genesets that are unique to a given state; and d) comparing a profile ofbone marrow derived isolated mesenchymal progenitor cell of unknownstate to the gene sets.

Additionally, the present invention provides a method for identifyingtherapeutic targets for treatment of hematopoietic function by: a)determining the median gene expression profile of bone marrow isolatedpluri-differentiated mesenchymal progenitor cells associated with eachdisease state of interest; b) identifying gene groups that areup-regulated, down regulated, and common to each disease state; and c)identifying gene sets that are unique to a given state.

The present invention also includes therapeutic compositions includingisolated pluri-differentiated mesenchymal progenitor cells and apharmaceutically acceptable carrier, wherein the pluri-differentiatedmesenchymal progenitor cells are present in an amount effective toenhance bone marrow engraftment in a mammal in need thereof; enhancehematopoietic progenitor cell engraftment in a mammal in need thereof;or treat GvHD in a mammal about to undergo bone marrow or organtransplantation or suffering from GvHD caused by bone marrow or organtransplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention can be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings. The following is a brief description of the drawings which arepresented only for the purposes of further illustrating the inventionand not for the purposes of limiting same. Referring to the drawingfigures, like reference numerals designate identical or correspondingelements throughout the several figures.

FIG. 1 is a photograph showing the phase contrast photomicrograph viewof a Dexter-type stromal cell monolayer reflecting on cellularcomplexity.

FIG. 2 is a photograph showing the percoll gradient centrifugationtechnique of the present invention that purifies the MPCs (2) in largequantities to greater than 95% purity.

FIG. 3 is a photograph showing the Wright-Giemsa staining of Dexter-typestromacell cultures depicting three morphologically identifiable cellpopulations, macrophages (5), hematopoietic cells (3), and themesenchymal progenitor cells (4) of the present invention.

FIGS. 4A-4H are showing a series of photomicrographs showing themorphologic and phenotypic characteristics of the MPCs of the presentinvention, as uncovered by staining for representative mesenchymal celllineage markers. The methods applied are shown in parentheses (FIG. 4A)Wright-Giemsa (Harleco stain using HMS Series Programmable SlideStainer, Carl Zeiss, Inc.). (FIG. 4B) Immunostain using anti-CD68antibody (Immunotech, Clone PG-M1; Vector, Vectastain Elite ABC Kit).(FIG. 4C) Immunostain using anti-CD45 antibody (Dako, Clone PD7/26 &2B11; ABC Kit). (FIG. 4D) Periodic acid-Schiff (Sigma). (FIG. 4E) NileRed (Sigma), counterstained with DAPI (Vector). (FIG. 4F) Alkalinephosphatase (Sigma Kit No. 85), counterstained with Nuclear Fast Red(Baker). (FIG. 4G) Immunostain using antibody to fibronectin(Immunotech, Clone 120.5; ABC Kit). (FIG. 4H) Immunostain usinganti-muscle actin antibody (Ventana, clone HUC 1-1; Ventana system usinga section of formalin-fixed, paraffin-embedded cell block, instead of acytospin). Appropriate positive controls and isotype-matched negativecontrols were employed to ascertain antibody staining-specificity. Allparts of figure as shown, except FIGS. 4E and 4H, have clearlyidentifiable built-in cell controls. The morphological features of thecells are listed in row 1 of Table 1.

FIG. 5 is a photograph which shows a transmission electron micrograph ofan MPC of the present invention bearing microvilli, irregular nucleus,and pools of glycogen (6) in the ectoplasm (×4,600).

FIGS. 6A-6M are photographs which show Northern blot analysis of bonemarrow stromal cell RNAs for expression of genes specific for multiplemesenchymal cell lineages. FIGS. 6A-6M represent different gene probesused for hybridization. The following outlines the sources of the geneprobes employed and the approximate sizes of the major transcriptsobserved (shown in parentheses): FIG. 6A: CD68 (Clone ID 3176179, GenomeSystems, Inc (GSI); 2-3 kb); FIG. 6B: Cathepsin B (Clone ID 2806166,GSI; 2-3 kb); FIG. 6C: GAPDH probe (generated using PCR primers from R&DSystems, Inc; ˜2 kb) hybridized to same blot as A and B; FIG. 6D:Adipsin (probe generated using PCR primers as described, Ref 20; 0.5-1kb); FIG. 6E: Osteoblast-specific cadherin-11 (Clone ID 434771, GSI; ˜3kb); FIG. 6F: Chondroitin sulfate proteoglycan 2 (Clone ID 1623237,GSI; >10 kb); FIG. 6G: Collagen type I alpha 1 (Clone ID 782235,GSI; >10 kb); FIG. 6H: Decorin (Clone ID 3820761, GSI; 2-3 kb); FIG. 6I:GAPDH probe hybridized to same blot as D-H; FIG. 6J: Fibronectin (CloneID 3553729, GSI; >10 kb); FIG. 6K: Caldesmon (Clone ID 1319608, GSI; ˜4kb); FIG. 6L: Transgelin (Clone ID 4049957, GSI; ˜1.5 kb); and FIG. 6M:GAPDH probe hybridized to same blot as J-L.

FIG. 7 is a photograph which shows RT-PCR analysis for expression ofrepresentative hematopoietic growth factors (G-CSF and SCF) andextracellular matrix receptors (ICAM-1, VCAM-1, and ALCAM) by the MPCsof the present invention.

FIG. 8 is a graph illustrating the comparison of the ability to supportin vitro hematopoiesis by the purified MPCs (heavy fraction representedby gray) of the present invention vs. unfractionated bone marrow stromalcells (represented by black).

FIGS. 9A-9B are graphs showing flow cytometric evidence of humanhematopoietic cell engraftment in a SCID mouse cotransplanted with theMPCs of the present invention. FIG. 9A shows CD45+/CD34+ progenitors inthe marrow. FIG. 9B shows CD45/CD34-mature hematopoietic cellscirculating in the blood.

FIGS. 10A-10H are photographs which show engraftment of humanhematopoietic cells in a SCID mouse cotransplanted with the purifiedmarrow MPCs of the present invention. FIG. 10A shows a serial section ofa mouse spleen stained with H & E. FIG. 10B shows a serial section of amouse spleen stained with immunoperoxidase stain for CD45. FIG. 10Cshows bone marrow stained for CD45. FIG. 10D shows a serial section ofthe mouse liver stained with H&E depicting involvement of periportalareas. FIG. 10E shows a serial section of the mouse stomach stained withH&E showing transmural infiltration. FIG. 10F shows a serial section ofthe mouse lung stained with H&E showing involvement of peribronchialarea. FIG. 10G shows a serial section of the mouse pancreas stained withH&E. FIG. 10H shows a serial section of the mouse paravertebral gangliastained with H&E.

FIG. 11A is a photomicrograph of a serial section of the spleen of anormal BALB/C mouse showing white pulp populated by darkly staininglymphocytes (H&E). FIG. 11B is a photomicrograph of the spleen of a SCIDmouse showing white pulp largely consisting of lightly staining stromalframework (H&E). FIG. 11C is a photomicrograph of the spleen of a SCIDmouse cotransplanted with human bone marrow MNC and the purified bonemarrow MPCs of the present invention showing homing (engraftment) ofhuman B cells to white pulp.

FIGS. 12A-12C are photographs which show Southern blotting data. FIG.12A shows that hybridization of sample DNA using a DNA probe specificfor human chromosome 17 alpha satellite DNA (p17H8) results in a 2.7 Kbband (7) (arrow; autoradiogram exposed for only 45 minutes). FIG. 12Bshows EcoR1 digest of thymic genomic DNA from SCID mice. FIG. 12C showsEcoR1 digest of lymph node genomic DNA from SCID mice.

FIGS. 13A-1, 13A-2, 13B-1, and 13B-2 show graphs comparing the survivalrate and engraftment of human hematopoietic cells in SCID micecotransplanted with the purified bone marrow MPCs of the presentinvention vs. unpurified bone marrow stromal cells. In the line graphsprovided (FIGS. 13A-1 and 13B-1) the line with diamonds represents MPCsand bone marrow mononuclear cells, squares represents bone marrowmononuclear cells only, triangles represents unfractionated bone marrowstromal cells, the Xs represent MPCs only, and the circles represent thecontrol. In the bar graphs (FIGS. 13A-2 and 13B-2), the gray barsrepresent mice that survived and the black bars represent mice withengraftment.

FIGS. 14A-14C are photographs which demonstrate apoptosis by TUNEL assayin organs of SCID mice that died after transplantation. FIG. 14A shows aserial section of the liver of the mouse that survived. FIG. 14B shows aserial section of the liver of the mouse that died. FIG. 14C shows aserial section of the spleen of the mouse that survived. FIG. 14D showsa serial section of the spleen of the mouse that died.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention provides isolated and purifiedmesenchymal progenitor cells that are pluri-differentiated. Alsoprovided by the present invention is a therapeutic composition includingan effective amount of isolated and purified pluri-differentiatedmesenchymal progenitor cells and a pharmaceutically acceptable carrier.

The present invention is premised upon the discovery that Dexter-typecultures contain stromal cells that co-express multiple message lineagemarkers. These pluri-differentiated cells are referred to by theinventor as mesenchymal progenitor cells (MPCs). Disclosed herein is aprocess for isolating and purifying MPCs from Dexter-type cultures.Purified MPCs provide a sufficiently defined system to permit detailedelucidation of the role of bone marrow in normal and leukemichematopoiesis. The present invention is also directed to various methodsfor using MPCs to enhance bone marrow transplantation, enhancehematopoietic progenitor cell engraftment, for diagnostic purposes, orfor the treatment of GvHD.

The term “pluri-differentiated” as used herein refers to cells that area single cell type co-expressing genes specific for multiple lineages.The term “pluri-potential” as used herein refers to cells that areundifferentiated and have the potential to be differentiated intodiscrete mesenchymal tissues.

Dexter type bone marrow cultures are a widely used and favorite mediumfor the study of hematopoiesis. Conventional wisdom has held that thestromal cells in Dexter-type cultures comprise a mixture of macrophages,hematopoietic cells, adipocytes, osteoblasts, fibroblasts, muscle cells,and endothelial cells. As a result of this perceived cellularcomplexity, research efforts over the last 23 years were not directed tocharacterizing or isolating the mesenchymal cells from the Dexter-typecultures.

Characterization of Cells. The exact cell types in Dexter cultures havebeen identified. No evidence was found for the existence of discretecellular populations, such as adipocytes, osteoblasts, fibroblasts,smooth muscle cells and endothelial cells, notwithstanding the abundanceof literature and wide spread belief (See, Liesveld, J. L. et al. Blood,1989, 73:1794; Sullivan, A. K. et al. Lab Invest, 1989, 60:667;Dorshlind, K. Ann Rev Immunol, 1990, 8:126; Perkins, S. and Fleischman,R. A. Blood, 1990, 75:620; Denkers, I. A. et al. Ann Hematol, 1992,64:210; Penn, P. E. et al. Blood, 1993, 81:1205; de Wynter, E. et al. JCell Sci, 1993, 106:761; Ferrajoli, A. et al. Stem Cells (Dayt), 1994,12:638; Clark, B. R. and Keating, A. Ann NY Acad Sci, 1995, 770:70;Wilkins, B. S. and Jones, D. B. Br J Haematol, 1995, 90:757; Gronthos,S. and Simmons, P. J. J Hematother, 1996, 5:15; Soligo, D. et al. Blood,94(Supplement 1 (Part 2 of 2)):168b, Abstract #3926, Forty 1^(st) AnnualMeeting of the American Society of Hematology, New Orleans, La., Dec.3-7, 1999; Dorheim, M-A. et al. J Cell Physiol, 1993, 154:317, Majumdar,M. K. et al. J Cell Physiol., 1998, 176:57, Prockop, D. J. Science,1997, 276:71, Taichman, R. S. and Emerson, S. G. J Exp Med, 1994,179:1677; Taichman, R. S. et al. Blood, 1996, 87:518; Verfaillie, C. M.in HEMATOLOGY: Basic Principles and Practice, R. Hoffman, et al., Eds.,Churchill Livingstone, New York, 2000, pp. 140-142, Henderson, A. J. etal. J Immunol, 1990, 145:423; Long, M. W. et al. J Clin Invest, 1990,86:1387; Simmons, P. J. et al. Prog Clin Biol Res, 1994, 389:271;Roecklein, B. A. and Torok-Storb, B. Blood, 1995, 85:997; Wineman, J. etal. Blood, 1996, 87:4082; Kelly, K. A. and Gimble, J. M. Endocrinology,1998, 139:2622; Hicok, K. C. et al. J Bone Miner Res, 1998, 13:205;Park, S. R. et al. Bone, 1999, 24:549; Dennis, J. E. et al. J Bone MinerRes, 1999, 14:700; and Torok-Storb, B. et al. Ann NY Acad Sci, 1999,872:164). Instead, the inventor determined that there are only threetypes of cells in Dexter-type cultures, namely, macrophages (˜35%),hematopoietic cells (˜5%), and a type applicant calls “nonhematopoieticcells” (˜60%) (FIG. 3, FIG. 4A, and Table 1).

Bone marrow mesenchymal cells, the nonhematopoietic cells in Dexter typecultures, possess distinctive features that have previously goneunrecognized. There is both direct visual (FIGS. 4A-4E and FIG. 5) andmolecular biological (FIG. 6) evidence to support the existence of thisunique cell type. These findings challenge the prevailing belief thatstromal cells derived from Dexter cultures comprise multiplesingly-differentiated mesenchymal cell types. Because Dexter culturesrepresent a primary cell culture system, and not a cell line, thesestudies indicate that cells in these primary cultures themselves arepluri-differentiated, which has been previously unsuspected. Thenonhematopoietic cells of the present invention (MPCs) simultaneouslyexpress marker genes specific for multiple mesenchymal cell lineages,including adipocytes, osteoblasts, fibroblasts and smooth muscle cells.

The MPCs in Dexter type cultures were characterized using a variety oftechniques. Cytospins were prepared using aliquots of unfractionatedcells for performance of various cytological, cytochemical andimmunocytochemical stains. Reactivity patterns of the bone marrowculture cells are outlined in Table 1. FIGS. 4A-4E illustratemorphologic and phenotypic characteristics, as uncovered by staining forrepresentative cell lineage markers.

Only rarely have investigators in this field taken the approach ofpreparing a cell suspension and staining cells on cytospins as was doneto characterize the cells of the present invention (Simmons et al.Nature, 1987, 328:429-432) and no other group has used this method toaddress the issue of pluri-differentiation by bone marrow stromal cells.Almost all of the published studies in the field, with a rare exception(Simmons et al. Nature, 1987, 328:429-432), conducted cytochemical andimmunocytochemical staining on layers of stromal cells grown toconfluence on coverslips. In this situation, the stromal cultures appearvery complex especially in the areas of hematopoietic activity, theso-called “cobblestones” with macrophages and hematopoietic cellsenmeshed in them. Macrophages and nonhematopoietic cells spreadthemselves and assume varied shapes when they adhere to and grow onplastic or glass that further contributes to the perceived heterogeneityand complexity. The complexity precludes a clear morphologicalvisualization of the nonhematopoietic cells and consequently interferedwith the determination of what percent of what cell type is positive forany given marker.

In terms of lineage markers, up to 100% of the nonhematopoietic cells orMPCs of the present invention expressed two fat cell markers (Nile Red(FIG. 4E) and Oil Red 0); an osteoblast marker (alkaline phosphatase(FIG. 4F)); and two fibroblast markers (fibronectin (FIG. 4G) andprolyl-4-hydroxylase). Greater than 85% of the MPCs were also positivefor a muscle marker, actin (FIG. 4H). There was no evidence ofexpression of endothelial cell differentiation, as judged byimmunohistochemical staining for CD34 and CD31.

In addition, the Dexter type stromal cells had not previously beensubjected to Periodic acid-Schiff (PAS) staining, which revealed astrong and uniform positivity by almost 100% of the MPCs studiedindicating the presence of large stores of glycogen (FIG. 4D). Thepresence of glycogen (6) was confirmed by electron microscopy (see FIG.5). In this respect, MPCs are reminiscent of the glycogen-ladenreticular cells in the developing bone marrow of human fetuses (observedby Chen, L-T. and Weiss, L. Blood, 1975, 46:389). Glycogen deposition isviewed to be a developmentally regulated process during morphogenesis(Ohshima, H. et al. Cell Tissue Res., 1999, 297:271).

The MPCs also exhibited cytoplasm compartmentalization into endoplasmand ectoplasm. This morphologic finding sheds light on their internalarchitecture because of correlation of restricted localization ofglycogen and smooth muscle actin to ectoplasm; and of acid phosphatase,alkaline phosphotase, Nile Red, Oil Red 0, fibronectin, andprolyl-4-hydrolase to endoplasm.

Additional sets of multiple mesenchymal lineage markers were assessed byNorthern blotting of unfractionated cells and purified MPCs to eliminateany observer bias that might be inherent in morphological assessment.FIGS. 6A-6M represent different gene probes used for hybridization. Thesources of the gene probes employed and the major transcripts observedare outlined in the brief description of the figures.

Compared to unfractionated cells, the purified nonhematopoietic cellsexpressed significantly higher levels of markers representing fat cells(adipsin, FIG. 6D); osteoblasts (osteoblast-specific cadherin-1,chondroitin sulfate, collagen type 1 and decorin, FIGS. 6E-6H);fibroblasts (fibronectin, FIG. 6J); and smooth muscle cells (caldesmonand transgelin, FIGS. 6K-6L).

Taken together, the morphologic, cytochemical, and immunocytochemicalresults (FIGS. 4A-4H and Table 1), and the Northern blotting data (FIGS.6A-6M) indicate that the nonhematopoietic stromal cells of the Dextercultures co-express markers specific for at least four differentmesenchymal cell lineages. Using a variety of techniques, applicant hasdemonstrated that the MPCs co-express multilineage mesenchymal cellphenotypes, and in this respect the multi- or pluri-differentiated MPCsare distinct from the pluri-potential, but undifferentiated, MSCs ofFriedenstein cultures (Prockop, Science, 1997, 276:71-74).

Isolation of MPCs. The nonhematopoietic cells of the present inventionwere purified from the macrophages, the dominant “contaminating” celltype, using a Percoll gradient method developed by applicant. MPCs werepurified by the following process: cells from a Dexter-type culture weretreated to obtain a cell suspension, the macrophages were removed, andthe cells were fractionated using discontinuous Percoll gradientcentrifugation (FIG. 2). The isolated MPCs were then collected andwashed.

The purity of the nonhematopoietic cells was demonstrated by a nearcomplete absence of two macrophage markers, CD68 and cathepsin B (asshown by Northern blotting data, FIGS. 6A and 6B). As a positivecontrol, bone marrow mononuclear cells rich in myelomonocytic cellsabundantly expressed CD68 (lanes 5 & 6, FIG. 6A). The Northern blotresults are consistent with a purity estimate of ˜95% (vs. 60% inunfractionated samples) based on morphology and immunocytochemicalstaining for CD68.

A purified source of MPCs is desirable for a number of reasons. Therelative ease with which large numbers of the MPCs can be purified andtheir distinctive phenotypic characteristics make them valuable targetsfor future investigations. Purified MPCs provide a sufficiently definedsystem to permit detailed elucidation of the role of bone marrow innormal and leukemic hematopoiesis in addition to aiding in bone marrowtransplantation.

Another major reason that purified cells are desirable is that Dextercultures also contain a significant percentage of highly immunogenicmacrophages that can cause onset of GvHD after transplantation. The MPCsof the present invention are purified to ˜95% free of macrophages andhematopoietic cells. Note the increased survival rate in SCID mice thatreceived purified MPCs versus those that received unfractionated bonemarrow stromal cells in FIGS. 13B-1 and 13B-2.

Enhancing Engraftment. The present invention also provides methods ofenhancing the engraftment of hematopoietic cells and of enhancing theengraftment of bone marrow. The hematopoietic support capacity of theDexter-type cultures has been repeatedly demonstrated by a number ofinvestigators. RT-PCR analysis showed that Dexter cultures andFriedenstein cultures expressed a similar pattern of cytokine and growthfactor mRNAs; yet, Dexter cultures were found to be more efficient thanFriedenstein cultures in achieving preservation of hematopoieticprogenitors (Majumdar et al. J. Cell. Physiol., 1998, 176:57-66). Thepluri-differentiated MPC is capable of supporting hematopoiesis, asshown by its ability to express representative hematopoietic growthfactors/cytokines, i.e., G-CSF and SCF as well as matrixreceptors/hematopoietic cell adhesion molecules, i.e., ICAM-1, VCAM-1and ALCAM (FIG. 7).

Clarification of the nature of the stromal cells and the ability topurify these cells makes it possible to use them as an adjuvant in bonemarrow transplantation following high-dose chemotherapy and radiationtherapy. These treatment modalities not only cause damage to thehematopoietic stem cells but also to the supportive stromal cells.However, because the bone marrow microenvironment is destroyed,hematopoietic progenitor cell engraftment is delayed until the stromalenvironment is restored. As a result, a critical aspect of the currentinvention is directed to the advantages of transplanting isolatedmesenchymal progenitor cells to accelerate the process of stromalreconstruction and ultimately bone marrow engraftment. The stromal cellspresent in the standard bone marrow transplant are not sufficient innumber and can be supplemented with the cultured MPCs of the presentinvention.

Yet another embodiment of the current invention provides the use of MPCtransplantation to major leukemic conditions, such as acute myeloidleukemia (AML), myelodysplastic syndromes (MDS), chronic myeloidleukemia (CML) and multiple myeloma (MM). This is based on applicant'sdetermination that bone marrow stromal cells in a leukemia patient arefunctionally and structurally defective, regardless of the damage causedby chemotherapy and radiation therapy. Such defects in bone marrowstromal cells are likely to aid and abet leukemia development.Alternatively, stromal cell defects could be secondarily induced bysurrounding leukemia cells, thus contributing to the loss ofhematopoietic support function of stromal cells and hematopoieticfailure, which is an invariable feature in leukemia. Regardless whetherthe observed stromal cell defects are primary or secondary to theleukemic process, by reason of their indisputable impact on normalhematopoiesis, these defects remain to be corrected to improve thehematopoietic function.

Stromal cells have never been carefully investigated in terms ofgenomics in view of the widespread belief that they represent aheterogeneous mixture of cell types. Tissue or cellular heterogeneitypresents a major challenge for the application of microarray technology.The purified stromal cells of the present invention represent a singlepluridifferentiated MPG which allows for genomic study of the stromalcells and the development of new, more objective diagnostic tools forpatients suffering from leukemia conditions.

The ability to purify culture-expanded MPCs from both normal individualsand patients afflicted with various leukemias also allows testing of thehematopoietic supportive role of MPCs in mice models. These systemsprovide an in vivo model in which to examine the role of human bonemarrow microenvironment in normal and leukemic hematopoiesis.

The Severe Combined Immunodeficiency Disease (SCID) mouse model is anideal system in which to investigate MPC function. Engraftment of humanhematopoietic progenitors in SCID mice has required eithercoadministration of exogenous human cytokines, or cotransplantation ofhuman bone marrow plugs or bone fragments. As disclosed herein MPCs area convenient, new source for human bone marrow stromal cells forenhancing transplantation that does not require cytokines, bonefragment, or marrow.

Unlike prior methods, the isolated MPCs of the present invention supporthuman hematopoiesis in the SCID mouse model as effectively as wholemarrow stroma. The transplantation of human marrow mononuclear cellscombined with purified MPCs results in dramatically vigorous engraftmentof human cells in spleen, bone marrow, liver, pancreas, lungs, stomach,and paravertebral neuronal ganglia of SCID mice (FIGS. 10A-10H and FIGS.11A-11C). By contrast, mice receiving human bone marrow mononuclearcells alone or MPCs alone expectedly showed no detectable evidence ofhuman hematopoietic cell engraftment (FIGS. 13A-1, 13A-2, 13B-1, and13B-2).

GvHD. The present invention also provides for a method of preventing ortreating GvHD. The highest mortality rate, FIGS. 13B-1 and 13B-2, wasobserved in mice receiving the unpurified whole marrow stroma and thebone marrow mononuclear cells. The increased mortality observed isrelated to the presence of highly immunogenic macrophages and consequentGvHD. The mice with the highest survival rate, shown in FIGS. 13A-1 and13A-2, were the mice receiving purified MPCs and bone marrow mononuclearcells.

Notably, there is discrete TUNEL-positive nuclei in the liver of theexpired mouse in FIG. 14B and complete absence of staining in the liverof the surviving mouse (see FIG. 14A). While some ill-defined globulesof staining are observed in the spleen of the mouse that survived, thenuclear integrity of most of the cells is well preserved suggestingminimal or no apoptosis (FIG. 14C). In contrast, the dead mouse spleen(FIG. 14D) showed extensive TUNEL positivity precluding accurateinterpretation. Control mouse liver and spleen showed results similar tothose of the mouse that survived.

The above results indicate that purified MPCs can support humanhematopoiesis in SCID mice as effectively as whole marrow stroma.Equally important is that the purified MPCs increased the survival rate.The evidence shows that the increased survival is due to a reduction inGvHD.

Allogeneic bone marrow transplantation is the preferred method oftreatment for a variety of malignant and genetic diseases of the bloodand blood forming cells. However, failure of hematopoietic cellengraftment can occur for a number of reasons. These include,microenvironmental defects as part of the underlying disease itself(e.g., aplastic anemia), and/or stromal cell damage caused bychemoradiotherapy and/or microenvironmental damage as part of GvHD whichis a dreaded complication following bone marrow transplantation. InGvHD, donor T cells present in the hematopoietic cell graft destroy hosttissues. GvHD can involve multiple organs such as skin, liver, GI systemetc. The current treatment modalities for graft failure or GvHD arecumbersome, costly and involve some form of immunosuppression. Stromalcell lesions either primary to the disease process or secondarilyinduced by allogeneic bone marrow transplantation play a prominent rolein the success or failure of the hematopoietic cell graft.Cotransplantation of MPC not only enhances hematopoietic cellengraftment but also prolongs the life of graft recipients by minimizingGvHD. Co-transplantation of healthy, culture-expanded MPC is a viableoption in these situations.

The human bone marrow used in the Dexter-type cultures of the presentinvention can be obtained from a number of different sources inaccordance with the procedures known in the art, including from plugs offemoral head cancerous bone pieces or from aspirated marrow. The cellsused in the Dexter culture can be autologous, from the tissue donor, orfrom other individuals.

Modes of administration of MPCs include, but are not limited to,systemic intravenous injection and injection directly to the intendedsite of activity. The MPCs can be administered by any convenient route,for example by infusion or bolus injection, and can be administeredtogether with other biologically active agents. Administration ispreferably systemic.

The methods of the present invention can be altered, particularly by (1)increasing or decreasing the time interval between administering MPCsand implanting the tissue, cells, or implanting the organs; (2)increasing or decreasing the amount of MPCs administered; (3) varyingthe number of MPC administrations; (4) varying the method of delivery ofthe MPCs; (5) or varying the source of MPCs.

The MPC preparations are used in an amount effective to promoteengraftment of hematopoietic progenitor cells or bone marrow cells; orfor the treatment or prevention of GvHD in the recipient. Thepharmaceutically effective amount for the purposes herein is thusdetermined by such considerations as are known in the art. In general,such amounts are typically at least 1×10⁴ MPCs per kg of body weight andmost generally need not be more than 7×10⁵ MPCs/kg.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of MPCs and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to McCoy's medium, saline, buffered saline,dextrose, water, and combinations thereof. The formulation should suitthe method of administration.

In one embodiment, the MPC preparation or composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition can alsoinclude a local anesthetic to ameliorate any pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a cryopreservedconcentrate in a hermetically sealed container such as an ampouleindicating the quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients can bemixed prior to administration.

The present invention paves the way for applications of mesenchymalprogenitor cells in the field of transplantation with respect tohematopoietic support, immunoregulation, and graft facilitation. MPCscan be used as a supporting cell type in bone marrow transplantation,particularly in diseases where defects in the hematopoietic stromalmicroenvironment are believed to prevail, such as aplastic anemia,myelofibrosis, and bone marrow failure following high dose chemotherapyand radiation therapy.

Diagnostic Applications. Another aspect of the invention provides amethod for diagnosing various disease states in mammals by identifyingnew diagnostic markers, specifically the classification and diagnosis ofleukemia. Prior to the present invention, stromal cells were notcarefully investigated in terms of genomics because of the widespreadbelief that they represent a heterogeneous mixture of cell types andcellular heterogeneity presents significant challenges for theapplication of genetic analysis such as microarray technology. Theisolated MPCs of the present invention represent a single cell type andallow for genomic study of the stromal cells.

Using the methods of the present invention, it has been determined thatbone marrow stromal cells in leukemia patients are functionally andstructurally defective regardless of the damage caused by chemotherapyand radiation therapy. Given the almost 25 year history and intenseinterest in bone marrow stromal cell cultures, previous documentation ofstromal cell abnormalities has been disappointingly low (Martinez &Martinez, Exp. Hematol, 1983, 11:522-526; Budak-Alpdogan et al. Am. JHematol, 1999, 62:212-220; Nagao et al. Blood, 1983, 61:589-592; Peledet al. Exp. Hematol, 1996, 24:728-737; Bhatia et al. Blood, 1995,85:3636-3645; Agarwal et al. Blood, 1995, 85:1306-1312; Diana et al.Blood, 2000, 96:357a). By identifying gene sets that are unique to agiven state, these differences in the stromal cells can be utilized fordiagnostic purposes.

In one embodiment of the invention, isolated MPCs from a patient areassayed for expression of a large number of genes. The gene expressionprofile is projected into a profile of gene set expression valuesaccording to the definition of gene sets. A reference databasecontaining a number of reference projected profiles is also created fromthe isolated MPCs of patients with known states, such as normal andvarious leukemic disease states. The projected profile is then comparedwith the reference database containing the reference projected profiles.If the projected profile of the patient matches best with the profile ofa particular disease state in the database, the patient is diagnosed ashaving such disease state. Various computer systems and software, seeExample 5, can be utilized for implementing the analytical methods ofthis invention and are apparent to one of skill in the art. Some ofthese software programs include Cluster & TreeView (Stanford),GeneCluster (MIT/Whitehead Institute), Array Explorer (SpotFire Inc) andGENESPRING (Silicon Genetics Inc) (for computer systems and software,see also U.S. Pat. No. 6,203,987).

The methods of the present invention can also be useful for monitoringthe progression of diseases and the effectiveness of treatments. Forexample, by comparing the projected profile prior to treatment with theprofile after treatment.

Therapeutic Applications. One aspect of the invention provides methodsfor therapeutic and drug discovery utilizing bone marrow derivedisolated mesenchymal progenitor cells. The present invention can beutilized to identify stromal cell genes that can be therapeutic targetsfor improvement of normal hematopoietic function, which is constantlycompromised, in leukemic patients. In one embodiment, gene sets aredefined using cluster analysis. The genes within a gene set areindicated as potentially co-regulated under the conditions of interest.Co-regulated genes are further explored as potentially being involved ina regulatory pathway. Identification of genes involved in a regulatorypathway provides useful information for designing and screening newdrugs.

Some embodiments of the invention employ gene set definition andprojection to identify drug action pathways. In one embodiment, theexpression changes of a large number of genes in response to theapplication of a drug are measured. The expression change profile isprojected into a gene set expression change profile. In some cases, eachof the gene sets represents one particular pathway with a definedbiological purpose. By examining the change of gene sets, the actionpathway can be deciphered. In some other cases, the expression changeprofile is compared with a database of projected profiles obtained byperturbing many different pathways. If the projected profile is similarto a projected profile derived from a known perturbation, the actionpathway of the drug is indicated as similar to the known perturbation.Identification of drug action pathways is useful for drug discovery.See, Stoughton and Friend, Methods for Identifying pathways of DrugAction, U.S. patent application Ser. No. 09/074,983.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

EXAMPLES

The examples presented in this experiment can be summarized as follows.The data disclosed herein demonstrate that Dexter cultures consist ofonly three cell types macrophages (˜35%), hematopoietic cells (˜5%), andnonhematopoietic cells (˜60%). Using a percoll gradient centrifugationtechnique, the nonhematopoietic mesenchymal progenitor cells wereisolated, free of macrophages and hematopoietic cells. A variety oftechniques were used to identify the isolated cells as amulti-differentiated mesenchymal cell lineage co-expressing genesspecific for multiple mesenchymal cell lineages including adipocytes,osteoblasts, fibroblasts and muscle cells.

Evidence that this multi- or pluri-differentiated mesenchymal progenitorcell is capable of supporting hematopoiesis is shown by the expressionof a number of hematopoietic growth factors and extracellular matrixreceptors. The SCID mouse experimental data provides evidence that sincethe MPCs can be purified to near homogeneity (95%) with relative ease,MPCs can be of value for enhancing engraftment of hematopoietic stemcells and bone marrow transplants. Additionally, increased survival ratein the SCID mouse model indicates that isolated MPCs can also be usefulfor the treatment of GvHD. An example of the administration of bonemarrow cells and MPCs to breast cancer patients treated withchemotherapy is also provided.

A stepwise genomics strategy and an example of the genomic changesobserved in leukemia associated MPCs is also provided. Cluster analysiswas performed to show gene expression patterns in isolated MPCs of anormal individual and individuals with different leukemic conditions.The approach presented provides the basis for a new more objective meansto diagnose patients suffering from leukemic conditions.

Example 1 Isolation and Characterization of MPCs from Dexter-Type BoneMarrow Stromal Cell Culture Systems

Bone Marrow Culture: Bone marrow samples were obtained from posteriorsuperior iliac crest under general anesthesia for standard marrowtransplantation. Marrow stromal cell cultures were set up using theresidual cells recovered from the filters of Fenwal Bone MarrowCollection System after complete filtration of the marrow samples. Thefilters were rinsed with phosphate-buffered saline without Ca²⁺ and Mg²⁺(PBS-CMF). The cell suspension was subjected to Ficoll gradientisolation of the mononuclear cells (bone marrow MNCs). The bone marrowMNCs were washed (×2) in PBS-CMF and suspended in McCoy's 5A with HEPESmedium containing 12.5% fetal bovine serum (FBS), 12.5% horse serum, 1μM/L hydrocortisone and 1% penicillin/streptomycin (for this studyMcCoy's complete medium) and cultured under standard stromal-cellculture conditions (FIG. 1) (Seshi et al. Blood, 1994, 83:2399; Gartneret al. Proc Natl Acad Sci USA, 1980, 77:4756). After two weeks,confluent stromal cell cultures were trypsinized (first passage),followed by splitting each T75 flask into two T150 flasks.

Morphologic and Phenotypic Characteristics of MPCs as Uncovered byStaining for Representative Mesenchymal Cell Lineage Markers: Two weeksafter the first passage (above), confluent stromal cells were againtrypsinized. Cytospins were prepared using aliquots of unfractionatedcells for performance of various cytological, cytochemical andimmunocytochemical stains.

Reactivity patterns of the bone marrow culture cells are outlined inTable 1. FIGS. 4A-4E illustrate morphologic and phenotypiccharacteristics, as uncovered by staining for representative celllineage markers. As illustrated in Table 1 and FIGS. 3 and 4A,Wright-Giemsa staining revealed three morphologically identifiable cellpopulations in Dexter type stromal cell cultures, macrophages,hematopoietic cells, and nonhematopoietic cells (labeled 4, 3, and 5,respectively).

The identity of macrophages was confirmed by immunostain using anti-CD68antibody (FIG. 4B) and cytochemical stains for acid phosphatase andSudan black. The identity of hematopoietic cells (including macrophages)was confirmed by immunostain using anti-CD45 antibody (FIG. 4C).

The remaining nonhematopoietic cells stained intensely positive forPeriodic acid-Schiff, which was diastase sensitive, signifying thepresence of large stores of glycogen (FIG. 4D). The presence of glycogen(6) was confirmed by electron microscopy (see FIG. 5). In this respect,MPCs are reminiscent of the glycogen-laden reticular cells in thedeveloping bone marrow of human fetuses (observed by Chen, L-T. andWeiss, L. Blood, 1975, 46:389). Glycogen deposition is viewed to be adevelopmentally regulated process during morphogenesis (Ohshima, H. etal. Cell Tissue Res., 1999, 297:271).

In terms of lineage markers, up to 100% of the nonhematopoietic cellsexpressed two fat cell markers (Nile Red (FIG. 4E) and Oil Red 0); anosteoblast marker (alkaline phosphatase (FIG. 4F)); and two fibroblastmarkers (fibronectin (FIG. 4G) and prolyl-4-hydroxylase). Greater than85% of the nonhematopoietic cells were also positive for a musclemarker, actin (FIG. 4H). There was no evidence of expression ofendothelial cell differentiation, as judged by immunohistochemicalstaining for CD34 and CD31 (data not shown).

The results indicate that the nonhematopoietic cells of the Dextercultures are in fact a single, pluri-differentiated cell typeco-expressing multiple mesenchymal cell lineage markers. Thepluri-differentiated mesenchymal progenitor cells reported here are tobe distinguished from the pluri-potential, but undifferentiated, MSCsthat are generated in the absence of hematopoietic cells, such as inFriedenstein-type cultures. TABLE 1 Reactivity patterns of bone marrowstromal cells based on cytological, cytochemical and immunocytochemicalstains*,*** Test Hematopoietic Mesenchymal Figure Utilized MacrophagesCells Progenitor Cells 1 3 and 4A Wright- Large cells Small cells Largecells with a Giemsa with a small with minimal relatively irregular(Harleco) round nucleus amount of nucleus & & foamy cytoplasm: cytoplasmcytoplasm: 5% of total compartmentalized 35% of total cells intoectoplasm and cells endoplasm: 60% of total cells 2 4D Periodic acid- 00 ˜100% MPCs: Schiff (PAS) staining restricted (Sigma) to ectoplasm in aring-like fashion; and completely abolished by diastase digestion 3 4CCD45 (Dako, 100% 100% HCs 0 PD7/26 & macrophages 2B11) (MΦ) 4 4B CD68100% MΦ 0 0 (Immunotech, clone PG-M1) 5 Sudan Black ˜100% MΦ 0 0 (Sigma)6 Acid 100% MΦ; 0 100% MPCs; phosphatase positive positive granules(Sigma Kit granules in moderate No. 387) packed amounts; stainingthroughout restricted to cytoplasm endoplasm 7 4E Nile Red 0 0 ˜100%MPCs: (Sigma) staining restricted to endoplasm 8 Oil Red O 0 0 ˜100%MPCs: (Sigma) staining restricted to endoplasm 9 4F Alkaline 0 0 ˜100%MPCs: phosphatase variable number of (Sigma Kit positive granules; No.85) staining restricted to endoplasm & plasma membrane** 10 4GFibronectin 0 0 ˜100% MPCs: (Immunotech, staining restricted clone120.5) to endoplasm 11 Prolyl-4- 0 0 ˜100% MPCs: hydroxylase staining(Dako, clone preferentially in 5B5) the endoplasm 12 4H Muscle actin 00 >85% MPCs: (Ventana, variable staining clone HUC 1- restricted to 1)ectoplasm*The lineages of the markers tested above are: 3, hematopoietic cellmarker; 4, 5 and 6, monocyte/macrophage markers; 7 and 8, adipocytemarkers; 9, osteoblast marker; 10 and 11, fibroblast markers; 12 musclemarker.**One earlier study (Simmons et al. Nature, 1987, 328: 429-432)interpreted the localization of alkaline phosphatase staining asconfined to the plasma membrane when in fact it is predominately presentwithin the endoplasm (compare FIG. 1C of this reference with FIG. 4F).***While well-accepted mesenchymal lineage markers were used, thesemarkers do not necessarily lend themselves to simultaneous assessment ofthe same cell. For example, muscle-specific# actin antibody worked only on formalin-fixed, paraffin embeddedmaterial, whereas stains like alkaline phosphatase, Oil # Red and NileRed are not anti-body based and involve varying fixing and stainingconditions. Thus, the evidence shows that close to 100% of members of amorphologically distinct population # express multiple lineage markersof interest.

Bone Marrow Mesenchymal Progenitor Cell (MPC) Purification: To furtherinvestigate the characteristics of the MPCs, the nonhematopoieticstromal cells were then purified from the macrophages (˜95% pure), thedominant “contaminating” cell type using the following method. Confluentmonolayers of stromal cells resulting from first passage, above, werewashed for three minutes in Ca²⁺/Mg²⁺ free Hanks' balanced saltsolution. Cells were incubated at room temperature for 45 minutes withintermittent mixing in serum-free McCoy's medium containing 10 mML-leucine methyl ester (LME, Sigma). LME is a lysosomotropic agent thatselectively kills and detaches macrophages. The detached macrophageswere removed by washing the monolayers twice in McCoy's complete medium,followed by trypsinization of the monolayers. The resulting single cellsuspensions were fractionated by discontinuous Percoll gradient (70%,50%, 30%, 20%, 10%) centrifugation at 800×G for 15 minutes at 4° C. in afixed angle rotor (Avanti-J25 Beckman centrifuge) (FIG. 2). Low-densitycells representing the macrophages resistant to detachment by LMEseparate as a band at the interface of serum and 10% Percoll and werediscarded (1). High-density nonhematopoietic cells representing MPCsform a layer in the region of 30-50% Percoll (2). These were collectedand washed twice by centrifugation through PBS-CMF. This protocol isconservatively expected to yield, >2.5×10⁶ MPCs per T-150 flask (i.e.,>50×10⁶ MPCs per batch of 20 flasks). The purity of these preparations,typically about 95%, was routinely monitored by Wright-Giemsa staining.

Northern Blotting Additional sets of multiple mesenchymal lineagemarkers were assessed by Northern blotting to eliminate any observerbias that might be inherent in morphological assessment. FIGS. 6A-6Mrepresent different gene probes used for hybridization. The sources ofthe gene probes employed and the major transcripts observed are outlinedin the brief description of the figures.

Total RNA was prepared by dissolving the high-density cell pellets inTrizol (Life-Technologies). Total RNA samples from unfractionatedstromal cells and BM MNCs were similarly prepared. The RNA samples wereelectrophoresed in a standard 1% agarose gel containing 2% formaldehydein MOPS/EDTA buffer and blotted onto Immobilon-Ny+membrane. Probes werelabeled using Prime-A-Gene Kit (Promega) and a.sup.32P dCTP (NEN).Hybridization was performed at 65° C. in modified Church's hybridizationsolution using 3×10⁶ counts/ml in 10 ml (Millipore, 1998).

In FIGS. 6A-6M, Northern blot analysis was performed side-by-side onfractionated stromal cells, nonhematopoietic cells freed of macrophages,and initial bone marrow mononuclear cell samples. Lanes 1 and 2represent total RNA samples (10 μg each) from unfractionated stromalcells (subjects S1 and S2, respectively). Lanes 3 and 4 represent totalRNA samples (10 μg each) from purified stromal MPCs (subjects S1 and S2,respectively). Lanes 5 and 6 represent total RNA samples (10 μg each)from bone marrow mononuclear cells, the starting cells for bone marrowcell cultures (subjects S3 and S4, respectively).

The large transcripts, especially of collagen (lane 1, FIG. 6G) andfibronectin (lane 1, FIG. 6J), in RNA extracted from unfractionatedstromal cells of subject 1 showed difficulty migrating into the gel.This observation correlates with the presence of an artifact ofunresolved positive material in lane 1, FIG. 6A. Since the RNA extractedfrom unfractionated stromal cells of the subject 2 did not present thisproblem (lane 2, FIG. 6G, FIG. 6J, and FIG. 6A), the observation doesnot impact on the overall interpretation of the results (see text). Thelineages of markers tested were: monocyte/macrophage markers, CD68 andcathepsin B; adipocyte marker, adipsin; osteoblast markers,osteoblast-specific cadherin-11, chondroitin sulfate proteoglycan 2,collagen type I alpha 1 and decorin; fibroblast marker, fibronectin;muscle markers, caldesmon and transgelin. Marker signals were normalizedto the amount of RNA loaded, which was based on densitometry of theGAPDH signals on the corresponding blot (Bio-Rad Model GS-700 ImagingDensitometer). Attenuation or enhancement of the marker signals in thepurified stromal MPCs (i.e., lanes 3 and 4) relative to unfractionatedstromal cells (i.e., lanes 1 and 2, respectively) is shown as fold Δ(decrease/increase) underneath the lanes 3 and 4; ND, means notdetermined.

The purity of the nonhematopoietic cells was demonstrated by a nearcomplete absence of two macrophage markers, CD68 and cathepsin B (asshown by Northern blotting data, FIGS. 6A and 6B). As a positivecontrol, bone marrow mononuclear cells rich in myelomonocytic cellsabundantly expressed CD68 (lanes 5 & 6, FIG. 6A). The Northern blotresults are consistent with a purity estimate of ˜95% (vs. 60% inunfractionated samples) based on morphology and immunocytochemicalstaining for CD68.

Compared to unfractionated cells, the purified nonhematopoietic cellsexpressed significantly higher levels of markers representing fat cells(adipsin, FIG. 6D); osteoblasts (osteoblast-specific cadherin-11,chondroitin sulfate, collagen type 1 and decorin, FIGS. 6E-6H);fibroblasts (fibronectin, FIG. 6J); and smooth muscle cells (caldesmonand transgelin, FIGS. 6K-6L).

No trace of osteoblast, fibroblast, or smooth muscle cell markers weredetected in the bone marrow mononuclear cells, suggesting a less thandetectable level of stromal cells or their precursors in bone marrowmononuclear cells. However, the fat cell marker, adipsin, was detectedin all samples including the bone marrow mononuclear cells.

Taken together, the morphologic, cytochemical and immunocytochemicalresults (FIGS. 4A-4H and Table 1), and the Northern blotting data (FIGS.6A-6M) indicate that the nonhematopoietic stromal cells of the Dextercultures co-express markers specific for at least four differentmesenchymal cell lineages.

This finding is especially intriguing because pluri-differentiation isoften a feature of neoplastic cells (Brambilia and Brambilia, Rev. Mal.Respir., 1986, 3:235; Pfeifer et al. Cancer Res., 1991, 51:3793-3801;Tolmay et al. Virchow's Arch, 1997, 430:209-212). However, a cytogeneticanalysis of the Percoll-gradient purified MPCs showed a normal GTWbanding pattern.

RT-PCR Analysis for Expression of Representative Hematopoietic GrowthFactors and Extracellular Matrix Receptors by MPCs: RT-PCR was conductedin a total reaction volume of 100 μl using 2 μg each of total RNA;corresponding primers; and a master mix of the PCR reagents. The RTconditions included sequential incubations at 42° C. for 15 minutes, 99°C. for five minutes, and 5° C. for five minutes. The PCR conditionsincluded: initial melting at 94° C. for four minutes; and cyclicalmelting at 94° C. for 45 seconds, annealing at 55° C. for 45 seconds andextension at 72° C. for 45 seconds with 34 cycles. PCR was terminatedafter final extension at 72° C. for ten minutes. Reaction products(G-CSF, SCF, each 25 μl; VCAM-1, ALCAM, each 50 μl; ICAM-1, 75 μl) wereconcentrated as necessary; electrophoresed along with a 100-bp DNAladder (GIBCO-BRL) in a standard agarose (1%) gel in TAE buffer; andstained with ethidium bromide.

PCR products, shown in FIG. 7 lanes labeled 1-2, were generated usingaliquots of the same RNA samples from purified stromal MPCs, as used forNorthern blotting shown under FIG. 6 lanes 3 and 4 respectively. Thegene transcripts amplified were as follows: G-CSF (granulocyte-colonystimulating factor); (Tachibana et al. Br. J Cancer, 1997, 76:163-174);SCF (stem cell factor, i.e., c-Kit ligand); (Saito et. al. Biochem,Biophys. Res. Commun., 1994, 13:1762-1769); ICAM-1 (intercellularadhesion molecule-1, CD54) and VCAM-1 (vascular cell adhesionmolecule-1, CD106) (primers from R&D); and ALCAM (activated leukocytecell adhesion molecule, CD166) (Bruder et al. J Bone Miner. Res., 1998,13:655-663).

The observed PCR products for G-CSF (600 bp, i.e., the top bright band)and ALCAM (175 bp) were significantly different from the expected sizes(278 bp; 372 bp, respectively). However, sequencing of the gel-purifiedPCR bands and subsequent BLAST search showed a 99-100% identity withrespective members. Attempts to detect c-Kit (i.e., SCF receptor) usingprimers as described (Saito et al. Biochem, Biophys. Res. Commun., 1994,13:1762-1769) amplified a PCR product of ˜300 bp with no homology toc-Kit (data not shown). The observed product sizes for SCF (˜730 bp);ICAM-1 (˜750 bp); and VCAM-1 (˜500 bp) were as expected.

As illustrated in FIG. 7, RT-PCR analysis showed that purified,multi-differentiated MPCs express both critical hematopoietic growthfactor/cytokines, such as G-CSF and SCF as well as matrixreceptors/hematopoietic cell adhesion molecules, i.e. ICAM-1, VCAM-1,and ALCAM.

Example 2 Comparison of the Ability to Support In Vitro Hematopoiesis byPurified MPCs Vs. Unfractionated Bone Marrow Stromal Cells

CD34+ positive cells (hematopoietic progenitor cells) were purified(Dynal kit) and cocultured with irradiated stromal monolayers for fiveweeks, followed by performance of standard colony assays forhematopoietic progenitors using methylcellulose medium supplemented withcolony stimulating factors (using MethoCult medium from Stem CellTechnologies, Inc, Canada). Unfractionated bone marrow stromal cells andpurified MPCs were prepared in the same manner as in Example 1. Data inFIG. 8 represents results from three experiments. Purified MPC providesincreased preservation of hematopoietic progenitor cells compared tounfractionated stromal cells.

Example 3 Animal Model for Enhanced Engraftment Capacity of MPCs

The Severe Combined Immunodeficiency Disease (SCID) mouse model is anideal system in which to investigate MPC function. Engraftment of humanhematopoietic progenitors in SCID mice requires either coadministrationof exogenous human cytokines, or cotransplantation of human bone marrowplugs or bone fragments.

There has been discovered a convenient, new source for human bone marrowstromal cells for enhancing transplantation that does not requirecytokines, bone fragment, or marrow. Unlike prior methods, the isolatedcells of the present invention support human hematopoiesis in the SCIDmouse model as effectively as whole marrow stroma. The transplantationof human marrow mononuclear cells combined with purified MPCs results indramatically vigorous engraftment of human cells in spleen, bone marrow,liver, pancreas, lungs, stomach, and paravertebral neuronal ganglia ofSCID mice. By contrast, mice receiving human bone marrow mononuclearcells alone or MPCs alone expectedly showed no detectable evidence ofhuman hematopoietic cell engraftment. Also notably, the mortality ratewas highest in mice that received unfractionated whole marrow stromawhereas purified MPC increased the survival rate which can be due toreduction in GvHD.

Transplantation of Human Cells in SCID Mice: Homozygous CB-17 scid/scidmice, six to eight weeks of age, were used. Lyophilized anti-asialo GM1rabbit antibody (Wako Chemicals) was suspended in 1 ml sterile ddH₂O,followed by pretreatment of mice with an IP injection of 20 ml (600 mg)ASGM1 antibody (to specifically deplete mouse macrophages and NK cells).Alternatively, one could use NOD/SCID mice lacking NK cell function,however, in light of highly promising preliminary results it was electedto continue use of scid/scid mice. The antibody treatment scheduleincluded four-hour pre-engraftment and every seven days thereafter forthe duration of the experiment. On the day of transplantation, the micewere irradiated with 200 or 300 cGy gamma-irradiation from a ¹³⁷CSsource. Approximately 2.5×10⁶ MPCs suspended in 0.5 ml McCoy's mediumand/or 25×10⁶ MNCs suspended in 0.2 ml were injected per mouse,intraperitoneally. Hematopoietic cell engraftment was assessed afterfive weeks by harvesting and analyzing representative hematopoietic andnonhematopoietic organs including blood, spleen, bone marrow (from twofemurs and tibia) from euthanized mice.

Flow Cytometric Evidence: FIGS. 9A and 9B are flow cytometric evidenceof human hemopoietic cells in a SCID mouse cotransplanted with marrowMPC. FIG. 9A shows the presence of CD45+/CD34+ progenitors in themarrow. FIG. 9B shows CD45/CD34-mature hematopoietic cells circulatingin the mouse's blood.

Photomicrographs of Cells: FIGS. 10A-10H shows engraftment of humanhematopoietic cells in a SCID mouse cotransplanted with the purifiedmarrow MPCs of the present invention. FIG. 10A shows a serial section ofa mouse spleen stained with H & E. FIG. 10B shows a serial section of amouse spleen stained with immunoperoxidase stain for CD45. FIG. 10Cshows bone marrow stained for CD45. FIG. 10D shows a serial section ofthe mouse liver stained with H&E depicting involvement of periportalareas. FIG. 10E shows a serial section of the mouse stomach stained withH&E showing transmural infiltration. FIG. 10F shows a serial section ofthe mouse lung stained with H&E showing involvement of peribronchialarea. FIG. 10G shows a serial section of the mouse pancreas stained withH&E. FIG. 10H shows a serial section of the mouse paravertebral gangliastained with H&E.

FIG. 11A is a photomicrograph of a serial section of the spleen of anormal BALB/C mouse showing white pulp populated by darkly staininglymphocytes (H&E). FIG. 11B is a photomicrograph of the spleen of a SCIDmouse showing white pulp largely consisting of lightly staining stromalframework (H&E). FIG. 11C is a photomicrograph of the spleen of a SCIDmouse cotransplanted with human bone marrow MNC and the purified bonemarrow MPCs of the present invention showing homing (engraftment) ofhuman B cells to white pulp.

Southern Blotting Data: Hybridization of sample DNA using a DNA probespecific for human chromosome 17 alpha satellite DNA (p17H8) showslinear signal intensity with a 2.7 Kb band (arrow; autoradiogram exposedfor only 45 minutes) (FIG. 12A). Lanes 1-10 contain human DNA starting1000 ng to 100 ng admixed with 0 ng 900 ng of mouse DNA, total amountDNA loaded in each lane being 1 ug, allowing construction of a standardcurve. The reported limit of detection with this technique is 0.05%human cells, which is more reliable than flow cytometry in detectingvery low levels of human cell engraftment.

FIG. 12B is a Southern blot of EcoR1 digest of thymic genomic DNA fromSCID mice. Lanes 1-5 were loaded with 500 through 100 ng human DNA.Lanes 6, 9-11 were loaded with DNA from mice which receivedunfractionated bone marrow stroma plus bone marrow mononuclear cells.Lanes 7, 8, 14, 15 were loaded with DNA from mice that received MPCsplus bone marrow mononuclear cells. Lanes 12, 13 were loaded with DNAfrom mice that received bone marrow mononuclear cells only. There isevidence of human cell engraftment in the mouse thymus in lanes 9 and 11and lanes 14 and 15 evidenced by the 2.7 Kb band. There was no evidenceof engraftment in mice that only received only bone marrow mononuclearcells, lanes 12 and 13.

FIG. 12C is EcoR1 digest of Lymph Node genomic DNA from SCID mice. Lanes1-5 were loaded with 500 through 100 ng human DNA. Lanes 6, 9-11 wereloaded with DNA from mice which received unfractionated bone marrowstroma plus bone marrow mononuclear cells. Lanes 7, 8, 14, 15 wereloaded with DNA from mice that received MPCs plus bone marrowmononuclear cells. Lanes 12, 13 were loaded with DNA from mice thatreceived bone marrow mononuclear cells only. While there was evidence ofengraftment of human cells in the mouse lymph nodes for mice thatreceived unfractioned bone marrow stromal cells and MPCs, there was noevidence of engraftment in mice that only received only bone marrowmononuclear cells, lanes 12 and 13.

Increased Survival and Evidence of MPC Effect on GvHD: FIGS. 13A-1,13A-2, 13B-1, and 13B-2 show graphs comparing the survival rate andengraftment of human hematopoietic cells in SCID mice cotransplantedwith the purified bone marrow MPCs of the present invention versusunpurified marrow stromal cells. Mice in FIGS. 13A-1 and 13A-2 received300 cGy irradiation dose and mice in FIGS. 13B-1 and 13B-2 received 200cGY of irradiation. FIGS. 13A-1, 13A-2, 13B-1, and 13B-2 show comparableengraftment of human hematopoietic cells in SCID mice cotransplantedwith purified MPCs versus unpurified bone marrow stromal cells and themarkedly enhanced survival of mice receiving purified MPCs. Notably, noengraftment was observed in mice receiving bone marrow mononuclear cellsalone.

The highest mortality rate, FIGS. 13B-1 and 13B-2, was observed in micereceiving the unpurified stromal cells and the bone marrow mononuclearcells. The increased mortality observed can be related to the presenceof highly immunogenic macrophages and consequent GvHD. The mice with thehighest survival rate, as shown in FIGS. 13A-1 and 13A-2, were the micereceiving purified MPCs and bone marrow mononuclear cells.

FIGS. 14A-14C demonstrate apoptosis by TUNEL assay in organs of SCIDmice that died after transplantation with human bone marrow mononuclearcells and unpurified bone marrow stromal cells. FIG. 14A shows a serialsection of the liver of the mouse that survived. FIG. 14B shows a serialsection of the liver of the mouse that died. FIG. 14C shows a serialsection of the spleen of the mouse that survived. FIG. 14D shows aserial section of the spleen of the mouse that died. Hematoxylincounterstain was applied to sections in FIGS. 14A and 14C. Methylgreencounterstain was applied to sections in FIGS. 14B and 14D.

Notably, there is discrete TUNEL-positive nuclei in the liver of theexpired mouse in FIG. 14B and complete absence of staining in the liverof the surviving mouse FIG. 14A. While some ill-defined globules ofstaining are observed in the spleen of the mouse that survived, thenuclear integrity of most of the cells is well preserved suggestingminimal or no apoptosis (FIG. 14C). By contrast, the dead mouse spleen(FIG. 14D) showed extensive TUNEL positivity precluding accurateinterpretation. Control mouse liver and spleen showed results similar tothose of the mouse that survived.

The size of the spleens from the mice that survived and the mice thatdied were compared. The dead mice were observed to have small andatrophic spleens correlating with lymphoid cell depletion and apoptosis.

The above results indicate that purified MPC can support humanhematopoiesis in SCID mice as effectively as whole marrow stroma.Equally important is that the purified MPCs increased the survival rate.Evidence suggests that the increased survival can be due to a reductionin GvHD.

Example 4 Administration of Bone Marrow Cells and Mesenchymal ProgenitorCells to Breast Cancer Patients Treated with Chemotherapy

A breast cancer patient undergoes a diagnostic posterior iliac crestbone marrow aspiration and biopsy using a local anesthetic. A smallportion (2 to 3 ml) of the aliquot (10 to 20 ml) of marrow is submittedfor routine histologic testing and determination of the presence oftumor cells using immunoperoxidase testing. The remainder of the cellsare Dexter cultured for MPCs as described above in Example 1.

The patient also undergoes placement of a pheresis central venouscatheter, and receives subcutaneous injections of G-CSF (filgrastin) 10μg/kg/day as described in Peters et al. (Peters et al. Blood, 1993,81:1709-1719; Chao et al. Blood, 1993, 81:2031-2035; Sheridan et al. TheLancet, 1.989, 2:891-895; Winter et al. Blood, 1993, 82:293a). G-CSFinjections begin at least three days before the first pheresis isinitiated. G-CSF therapy is withheld if the white blood cell count risesabove 40,000 μL and is resumed once the white blood cell count drops toless than 20,000 μL.

If the patient is receiving only G-CSF as the vehicle for “mobilization”of peripheral blood progenitor cells, the patient must not have receivedchemotherapy within four weeks of the planned pheresis. If the patienthas received both conventional chemotherapy and G-CSF treatment formobilization., the patient must not have received chemotherapy withinten days of the planned pheresis, and the white blood cell count must beat least 800 μL and the platelet count at least 30,000 μL.

Daily pheresis procedures are performed using a Cobe Spectra instrument(Cobe, Lakewood, Colo.), and each cellular collection is cryopreservedusing a controlled-rate liquid nitrogen freezer, until at least 15×10⁸mononuclear cells/kg are collected (Lazarus et al. Bone MarrowTransplant, 1991, 7:241-246). Each peripheral blood progenitor cell isprocessed and cryopreserved according to previously published techniques(Lazarus et al. J Clin, Oncol., 1992, 10:1682-1689; Lazarus et al. BoneMarrow Transplant, 1991, 7:241-246).

Eight days before the patient is infused with the autologous peripheralblood progenitor cells, the patient receives chemotherapy over a periodof 96 hours (four days), with the following chemotherapy agents: 1)Cyclophosphamide in a total dosage of 6 g/m² (1.5 g/m 2/day for fourdays) is given via continuous intravenous infusion at 500 mg/m² in 1,000ml normal saline every eight hours; 2) Thiotepa in a total dosage of 500mg/m²/day for four days) is given via continuous intravenous infusion at125 mg/m² in 1,000 ml normal saline every 24 hours; and 3) Carboplatinin a total dosage of 1,800 mg/m² (200 mg/m²/day for four days) is givenvia continuous intravenous infusion at 200 mg/m² in 1,000 ml of 5%dextrose in water every 24 hours.

The patient also receives 500 mg of Mesna in 50 ml normal saline IV over15 minutes every four hours for six days (144 hours), beginning with thefirst dose of cyclophosphamide.

At least 72 hours after the completion of the chemotherapy, the MPCs areharvested from the Dexter culture(s). MPCs are collected and purified asdescribed in Example 1. Cells are resuspended at approximately 10⁶cells/ml, and injected slowly intravenously over 15 minutes to provide atotal dosage of from 10 to about 5×10⁶ cells.

MPCs can also be frozen and thawed to use when needed. For example,unfractionated cells from a Dexter culture are frozen. Upon thawing thecells are plated for about two days. The MPCs are then purified as inExample 1 above. The MPCs are then replated with serum or in a serumfree media and can remain stable for up to six days.

The day after the patient receives the MPCs, the frozen autologousperipheral blood progenitor cells are removed from the liquid nitrogenrefrigerator, transported to the patient in liquid nitrogen, submersedin a 37° C. to 40° C. sterile water bath, and infused rapidlyintravenously without additional filtering or washing steps. GM-CSF inan amount of 250 μg/m² then is given as a daily subcutaneous injection,beginning three hours after completion of the autologous bloodprogenitor cell infusion. The GM-CSF is given daily until the peripheralblood neutrophil count exceeds 1,000/μL for three consecutive days.

Example 5 Genomic Changes Observed in Leukemia Associated MPCs

The following is one example of how normal hematopoiesis might becompromised in leukemic conditions. The cellular interactions thatunderlie leukemic bone marrow involve stromal cells, leukemia/lymphomacells, and normal hematopoietic progenitors (including those ofmyelopoiesis, erythropoiesis and megakaryocytopoiesis). In addition todisplacing normal hematopoietic cells, the leukemia/lymphoma cells canpotentially cause direct damage to the hematopoietic supportive stromalcells by inducing unwanted gene expression profiles and adverselyaffecting the normal hematopoiesis. The cellular interactions can beschematized as:

The point of this scheme is that regardless of whether stromal celllesions are primary or secondary to leukemogenesis, the normalhematopoietic function is invariably compromised in leukemic conditions,though different leukemias affect myelopoiesis, erythropoiesis andmegakaryocytopoiesis differentially. Contrary to the prevailing notion(see Marini, F. et al., “Mesenchymal Stem Cells from Patients withChronic Myelogenous Leukemia Patients can be Transduced with Common GeneTransfer Vectors at High Efficiency, and are Genotypically Normal”,42^(nd) Annual Meeting of the American Society of Hematology, Dec. 1-5,2000 Poster #665), there has been observed extensive and striking geneexpression changes in leukemia-associated bone marrow MPCs by usinghigh-resolution genomics. Therefore, one embodiment of the presentinvention is to use transplantation of tissue-culture expanded, purifiednormal MPCs to improve granulopoiesis, erythropoiesis andthrombopoiesis, in for example MDS (most of MDS patients do not die fromblast transformation but from complications related to cytopenias, i.e.,hematopoietic failure).

The studies targeted acute myeloid leukemia (AML), chronic myeloidleukemia (CML) and multiple myeloma (MM), one case of each. The AMLpatient was a 57 year-old woman with 52% myeloblasts in the bone marrowwith immunophenotype confirmed by flow cytometry and a karyotypicabnormality of 45, XX, −7(6)/46, XX [6]. Together with morphology, thediagnosis was AML arising in a background of myelodysplasia. The CMLpatient was a 35 year-old man with 2% blasts in the bone marrow andkaryotypic abnormalities of Philadelphia chromosome and BCR/ABL generearrangement. Together with morphology, the diagnosis was CML inchronic phase. The MM patient was a 61 year-old woman with a IgAmyeloma. The serum IgA level was 2.4 g/dl and the marrow plasma cellcount was 37%. None of the patients was treated prior to obtainingmarrow samples used in this study, to avoid any therapy-induced changescomplicating the disease-associated changes.

The leukemic samples consisted of marrow aspirates that remained unusedafter clinical diagnostic studies were preformed. A bone marrow sampleobtained from an adult healthy male who had consented to donate bonemarrow for standard marrow transplantation was simultaneously studied.The normal bone marrow sample consisted of residual cells recovered fromthe filters after complete filtration of the marrow sample. Setting upof Dexter-type stromal cell cultures and isolation of MPC were asdescribed in Example 1. The normal stromal cells were studied withoutand after stimulation with TNFα because TNFα (and IL-4) are regarded asnegative regulators of hematopoiesis. Notably these cytokines,especially TNFα, are elevated in marrow plasma of patients withmyelodysplastic syndromes (MDS), the clinical hallmarks of which areanemia, leukopenia and thrombocytopenia (i.e., pancytopenia). TNFα andIL-4 are considered possible mediators of hematopoietic dysregulationtypical of MDS.

A Stepwise Genomics Strategy Encompassed: Preparation of total RNA fromMPC samples→generation of cDNA→preparation of ds DNA→in vitrotranscription into cRNA→fragmentation of cRNA→hybridization of targetRNA to a microarray of known genes (Affymetrix genechip containing DNAfrom 12,000 known human genes, e.g., U95A oligonucleotidemicroarray)→analysis of differentially expressed genes using anappropriate software (GENESPRING) to discern the patterns of geneexpression or genomic signatures by a given MPC type.

Cluster Analysis Showing Gene Expression Patterns in Bone Marrow MPCIsolated from a Normal Individual and Patients with Different LeukemicConditions: Genes with correlated expression across bone marrow MPCtypes: GENESPRING was used for cluster analysis. Prior to application ofan agglomerative hierarchical clustering algorithm, microarray signalswere normalized across experiments (i.e., from one MPC type to another)making the median value of all of measurements unity, so differentexperiments are comparable to one another. The signals were alsonormalized across genes in order to remove the differing intensitysignals from multiple experimental readings. Genes that are inactiveacross all samples were eliminated from analysis. Notably, 7398 genesout of 12,626 genes (present on the Affymetrix genechip used) passed thefilter of a normalized signal intensity of at least 0.1 across at leastone of the five experiments performed. Cluster analysis was performedwith standard correlation (same as Pearson correlation around zero) asthe distance metric, a separation ratio of 0.5 and a minimum distance of0.001 as provided by the software application. A closer relationshipbetween CML- and MM-associated MPCs was observed, which in turn arerelated to AML-associated MPC, thus transforming global patterns of geneexpression into potentially meaningful relationships.

Two-dimensional cluster analysis of tissue vs. gene expression vectors:A gene tree was constructed. Genes cluster near each other on the “genetree” if they exhibit a strong correlation across MPC experiments andMPC tree branches move near each other if they exhibit a similar geneexpression profile. The data indicated that the two-way clusteringreadjusted the location of a number of genes resulting in accentuationof genomic signatures of each cell type. Investigators can usefullycatalog genes composing any unique or signature cluster of interest bycreating a gene list and disclosing their identities.

Self-organizing Map (SOM) Clusters (6×5) Show Differential GeneExpression in Bone Marrow MPC Isolated from Different HematopoieticConditions: Generation of SOM clusters involved prior normalization andfiltering of the data. SOM algorithm was applied as provided byGENESPRING. Visualization of SOM clusters in combination withhierarchical clustering (i.e., MPC tree) revealed correlated meaningfulpatterns of gene expression. Predicated on the basis of SOM operatingprinciple, the related SOM clusters tend to be located physically closeto each other. For example, the juxtaposition of the SOM clusters withthe common denominator containing genes that are up-regulated inAML/MDS-associated MPC. Whole or part of any SOM cluster can be selectedto make a gene list providing the identities of the genes involved.

Genes Highly Expressed in Normal MPC but Absent or Minimally Expressedin Leukemia-associated MPC: Lists of genes that are down-regulated inleukemia-associated MPC (AMUMDS, CML and MM) were created in comparisonto normal MPC. A Venn diagram was made using these three gene lists.GENESPRING allows creation of sublists of genes corresponding to union,intersection and exclusion. Transcriptional profiles of any of thesesublists of genes can be visualized across MPC samples of interest. Thefollowing is one such sublist of genes containing genes that are highlyexpressed in normal MPC and down-regulated in leukemia-associated MPCsrevealing the identity of the subset of genes of interest: putative,wg66h09.x1 Soares Homo sapiens cDNA clone, Homo sapiens mRNA forCMP-N-acetylneuraminic acid hydroxylase, Homo sapiens cDNA cloneDKFZp586G0421 (symptom: hutel), Human mRNA for histone H1x, Putativemonocarboxylate transporter Homo sapiens gene for LD78 alpha precursor,Interacts with SH3 proteins; similar to c-cbl proto-oncogene product,wg82b12.x1 Soares Homo sapiens cDNA clone, Human atrial natriureticpeptide clearance receptor (ANP C-receptor) mRNA, Human 71 kDa 2′5′oligoadenylate synthetase (p69 2-5A synthetase) mRNA, Homo sapienshMmTRA1 b mRNA, Human GOS2 protein gene, Preproenkephalin, Humanguanylate binding protein isoform I (GBP-2) mRNA, Human gene forhepatitis C associated microtubular aggregate protein p44, 17-kDaprotein, Human insulin-like growth factor binding protein 5 (IGFBP5)mRNA, GS3686, Human monoamine oxidase B (MAOB) mRNA, Insulin-like growthfactor 11 precursor, Human insulin-like growth factor binding protein 5(IGFBP5) mRNA, Similar to ribosomal protein L21, X-linked mentalretardation candidate gene, and Homo sapiens mRNA; cDNA DKFZp434A202.

Genes not Expressed in Normal MPC but Highly Expressed inLeukemia-associated MPC: Lists of genes that are up-regulated (insteadof down-regulated) in leukemia-associated MPCs (AML/MDS, CML and MM)were created in comparison to normal MPC and a Venn diagram was made.The following is one such sublist of genes containing genes that areinactive in normal MPC but up-regulated in leukemia-associated MPCsrevealing the identity of the subset of genes of interest:Beta-tropomyosin, Homo sapiens clone 24659 mRNA sequence, Human mRNA forDNA helicase Q1, OSF; contains SH3 domain and ankyrin repeat, ym22b12.r1Soares infant brain 1 NIB Homo sapiens cDNA clone, Human mRNA forpre-mRNA splicing factor SRp20, Human mRNA for golgialpha-mannosidasell, OSF-2os, Homo sapiens gene for Proline synthetase,hk02952 cDNA clone for KIAA0683, wi24g10.x1 Homo sapiens cDNA clone,Lysosomal enzyme; deficient in Sanfilippo B syndrome, CTP synthetase (AA1-591), WD repeat protein; similar to petunia AN11, Human mRNA for5′-terminal region of UMK, complete cds, Homo sapiens chemokine exodus-imRNA, complete cds, Human GPI-H mRNA, complete cds, Homo sapiens mRNAencoding RAMP1, Transforming growth factor-beta-2 precursor, and Homosapiens mRNA for KIAA0763 protein.

Visualizing Expression of Phenotypically & Functionally Relevant GenesAcross Samples of Normal & Disease-associated BM MPC: AlthoughGENESPRING is a highly flexible and user-friendly software application,it lacks the facility to create functionally relevant gene listscontaining user-defined key words. This limitation was overcome bydevising the following method via MICROSOFT EXCEL. A stepwise protocolto create such a gene list using EXCEL includes: Open the annotatedmicroarray genome file (e.g., Affymetrix U95A) in EXCEL→select thecolumn with gene names select Data from pull-down menu→Filter→AutoFilterCustom enter key words (e.g., cell adhesion or cell cycle)→OK generatesa new EXCEL worksheet with the list of genes containing the key words.Copy and paste the list of genes containing the key words intoGENESPRING and save the gene list with a meaningful name. Twenty-two(22) such functionally relevant gene lists (Table 2) were created.

The resulting approach is a simple and powerful way to peer into theexpression profiles of focused sets of functionally relevant genesacross samples of interest. For example, the human vascular celladhesion molecule-I (VCAM-1) gene is completely down-regulated inAML/MDS and the human insulin-like growth factor binding protein(hIGFBP1) gene is up-regulated in AML compared to all other samples.Similarly, Homo sapiens gene for LD78 alpha precursor is down-regulatedin all of leukemia-associated MPCs. Finally, the lineage markers CD45and CD68 are essentially absent from the leukemia-associated MPCsattesting to the high degree of purity achieved by the samplepreparation technique of the present invention.

Results

The genomic changes observed in leukemia-associated MPCs are striking.As shown in Table 2, the changes (up-regulation and/or down-regulation)involved hundreds of genes. These changes were most dramatic in MPCassociated with AML arising in a background of MDS and involved multipleclasses of genes (Tables 1-2). Expectedly, the TNFα-induced changes wereextensive. Given the high level of purity of MPC preparations, theenormous genomic changes observed are reflective of the underlyingpathologic lesions in the MPCs themselves (and not due to thecontaminating leukemic cells and/or macrophages). These studies stronglysupport the hypothesis that stromal cells in a leukemic patient arefunctionally defective and therefore purified MPCs are of value inrestoring the loss of hematopoietic function in leukemic patients. TABLE2 Magnitude of global gene expression changes in leukemia-associated andTNFa-stimulated MPCs in comparison to normal MPC AML/MDS MPC CML MPC MMMPC TNFa MPC # of genes 234 112 108 279 up-regulated # of genes 379 208251 164 down-regulated

TABLE 3 Functional classes of genes analyzed across normal and leukemia-associated MPCs Annexins (14) Caspases & apoptosis-related transcripts(33) Cadherins (50) Calmodulins/calmodulin-dependent kinases (25) Celladhesion molecules (20) Cathepsins (19) Collagens (71) Cell divisioncycle-related transcripts (36) Cytokines (19) Epidermal growth factorsand related transcripts (22) Fibroblast growth factors (21) Fibronectins(6) Galectins (6) Growth factors (136) IGF system (24)Interleukins/receptors (76) Integrins/disintegrins (70) Lineage-relatedmarkers (19) Laminins (13) Platelet-derived growth factors & receptors(12) TNF alpha-related transcripts (29) TGF beta-related transcripts(25)

The gene lists in Table 3 were created as described above and analyzedusing GENESPRING. The numerical value in parenthesis refers to thenumber of transcripts in the corresponding class of genes analyzed.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

The preceding descriptions of the invention are merely illustrative andshould not be considered as limiting the scope of the invention in anyway. From the foregoing description, one of ordinary skill in the artcan easily ascertain the essential characteristics of the instantinvention, and without departing from the spirit and scope thereof, canmake various changes and/or modifications of the inventions to adapt itto various usages and conditions. As such, these changes and/ormodifications are properly, equitably, and intended to be, within thefull range of equivalence of the following claims.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A composition of matter comprising an isolated mesenchymal progenitorcell that is pluri-differentiated.
 2. The composition of claim 1,wherein said composition of matter is a therapeutic compositioncomprising isolated pluri-differentiated mesenchymal progenitor cellsand a pharmaceutically acceptable carrier.
 3. A method for purifyingpluri-differentiated mesenchymal progenitor cells comprising the stepsof: a) providing a cell culture preparation by the Dexter method; b)treating the cells to obtain a cell suspension; c) removing macrophages;d) fractionating the cells; and e) collecting the fraction ofpluri-differentiated mesenchymal progenitor cells.
 4. A method forenhancing bone marrow engraftment in a mammal in need thereof,comprising administering isolated pluri-differentiated mesenchymalprogenitor cells and a bone marrow graft to the mammal, wherein theisolated pluri-differentiated mesenchymal progenitor cells areadministered in an amount effective to promote engraftment of the bonemarrow in the mammal.
 5. The method according to claim 4, wherein saidadministering includes intravenously injecting or directly injecting theisolated pluri-differentiated mesenchymal progenitor cells to the siteof intended activity.
 6. The method of claim 4, wherein the isolatedpluri-differentiated mesenchymal progenitor cells are administered in acell suspension also containing bone marrow graft cells.
 7. The methodaccording to claim 4, further including administering the isolatedpluri-differentiated mesenchymal progenitor cells in a cell suspensionalso containing bone marrow graft cells.
 8. A method for enhancingengraftment of hematopoietic progenitor cells in a mammal in needthereof, comprising administering isolated pluri-differentiatedmesenchymal progenitor cells and hematopoietic progenitor cells to themammal, wherein the isolated pluri-differentiated mesenchymal progenitorcells are administered in an amount effective to promote engraftment ofthe hematopoietic progenitor cells in the mammal.
 9. The method of claim8, wherein the isolated pluri-differentiated mesenchymal progenitorcells are administered by intravenous injection or by injecting directlyto the site of intended activity.
 10. The method of claim 8, wherein theisolated pluri-differentiated mesenchymal progenitor cells areadministered prior to administration of the hematopoietic progenitorcells.
 11. The method of claim 8, wherein the isolatedpluri-differentiated mesenchymal progenitor cells are introduced in acell suspension also containing hematopoietic progenitor cells.
 12. Amethod for treating graft-versus-host disease (GvHD) in a mammal aboutto undergo bone marrow or organ transplantation or suffering from GvHDcaused by bone marrow or organ transplantation, comprising administeringto the mammal an effective amount of isolated pluri-differentiatedmesenchymal progenitor cells.
 13. The method according to claim 12,wherein the mammal is one about to undergo allogeneic bone marrowtransplantation or is suffering from GvHD caused by allogeneic bonemarrow transplantation.
 14. The method according to claim 12, furthercomprising administering immunosuppressive drugs to the mammal.
 15. Themethod according to claim 12, wherein the isolated pluri-differentiatedmesenchymal progenitor cells are administered by intravenous injection.16. The method according to claim 12, wherein the isolatedpluri-differentiated mesenchymal progenitor cells are administered tothe mammal prior to bone marrow or organ transplantation.
 17. A methodfor diagnosing a disease state comprising the steps of: a) establishinggene expression patterns of normal state bone marrow derived isolatedpluri-differentiated mesenchymal progenitor cells; b) establishing geneexpression patterns of various leukemic state bone marrow derivedisolated pluri-differentiated mesenchymal progenitor cells; c)identifying gene sets that are unique to a given state; and d) comparinga profile of bone marrow derived isolated mesenchymal progenitor cell ofunknown state to said gene sets.
 18. A method for identifyingtherapeutic targets for treatment of hematopoietic function comprisingthe steps of: a) determining the median gene expression profile of bonemarrow isolated pluri-differentiated mesenchymal progenitor cellsassociated with each disease state of interest; b) identifying genegroups that are up-regulated, down-regulated, and common to each diseasestate; and c) identifying gene sets that are unique to a given state.